Allogeneic islet transplantation can be an effective treatment for end-stage type 1 diabetes (T1D) patients who have severe life-threatening complications. Unfortunately, the use of this promising procedure is limited due to a severe shortage of pancreata from human cadavers. Therefore, there is a critical need for innovative methods to grow and differentiate functional, insulin expressing cells in the laboratory that are suitable for transplant into diabetic patients. Our long-term goal is to develop novel methods for generating large numbers of functional, insulin-expressing cells for transplant. Because embryonic stem cell products have associated cancer risks, we have developed two innovative colony assays for analyzing the ability of single adult pancreas cells to self-renew, expand and differentiate into insulin producing cells. Therefore, our work directly addresses whether progenitors are present in the adult pancreas, which is a controversial question in pancreatic cell biology. For a definitive answer, assays capable of defining differentiation and self-renewal abilities at the single cell level with precision and ease were required, but until now, have not been available. Using our unique tools, we have discovered that adult murine pancreata do contain a small, sub-population of cells that can form colonies and differentiate into insulin+ cels in vitro. We have named these cells pancreatic colony-forming units (PCFUs), and by manipulating certain cell signaling pathways, we will determine how these PCFUs can self-renew and be induced to differentiate and further expand to generate the number of insulin+ cells needed for transplant to correct hyperglycemia in T1D mice. Critically, we now have preliminary data indicating that we can also isolate human PCFUs from pancreata that come from cadavers that have had their islets removed for transplant. We will adapt the strategies used for murine pancreatic cell to characterize these human PCFUs and develop methods for their enrichment and differentiation into functional insulin+ cells suitable for transplant. The proposed research is innovative because it represents a substantive departure from the current paradigm; it uses colony-forming assays of single cells to delineate the differentiation and expansion properties of adult pancreatic cells in a quantitative way. It is significant because it addresses the critical shortage of islet beta cells for transplants, and the need for safer cell products by using adult pancreas rather than embryonic stem cells. The results will have a positive impact on research aimed at generating glucose-responsive, insulin-secreting cells for T1D cell replacement therapy by identifying the adult pancreas as a viable cell source.